1. Field of the Invention
The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array, and in particular an Aβ1-6 peptide-VLP-composition. More specifically, the invention provides a composition comprising a virus-like particle and at least one Aβ1-6 peptide bound thereto. The invention also provides a process for producing the conjugates and the ordered and repetitive arrays, respectively. The compositions of the invention are useful in the production of vaccines for the treatment of Alzheimer's disease and as a pharmaccine to prevent or cure Alzheimer's disease and to efficiently induce immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.
2. Related Art
Alzheimer's disease (AD) is the most common cause of dementia among the elderly (age 65 and older) and a serious burden for public health. For example, 4 million people are reported to suffer from the disease in the United Sates of America. The incidence of the disease is expected to increase as the population ages.
The main pathological signs of Alzheimer's disease are age-related changes in behaviour, deposition of β-amyloid into insoluble plaques, called the neuritic plaques or AD plaques, neurofibrillary tangles composed of tau protein within neurons, and loss of neurons throughout the forebrain. In addition to the late onset AD, which occurs in old age (65 years and more), there is an early onset AD, familial AD (FAD) occurring between age 35 and 60. The pathological abnormalities of AD are more widespread, severe and occur earlier in FAD than in late onset or sporadic AD. Mutations in the APP gene, the presenilin 1 and the presenilin 2 genes have been correlated with FAD.
As indicated, one of the key events in Alzheimer's Disease (AD) is the deposition of amyloid as insoluble fibrous masses (amyloidogenesis) resulting in extracellular neuritic plaques and deposits around the walls of cerebral blood vessels (for review see Selkoe, D. J. (1999) Nature. 399, A23-31). The major constituent of the neuritic plaques and congophilic angiopathy is amyloid β (Aβ), although these deposits also contain other proteins such as glycosaminoglycans and apolipoproteins. Aβ is proteolytically cleaved from a much larger glycoprotein known as Amyloid Precursor Protein (APP), which comprises isoforms of 695-770 amino acids with a single hydrophobic transmembrane region. Aβ forms a group of peptides up to 43 amino acids in length showing considerable amino- and carboxy-terminal heterogeneity (truncation) as well as modifications (Roher, A. E., Palmer, K. C., Chau, V., & Ball, M. J. (1988) J. Cell Biol. 107, 2703-2716. Roher, A. E., Palmer, K. C., Yurewicz, E. C., Ball, M. J., & Greenberg, B. D. (1993) J. Neurochem. 61, 1916-1926). Prominent isoforms are Aβ1-40 and 1-42. It has a high propensity to form β-sheets aggregating into fibrils, which ultimately leads to the amyloid.
Aβ peptide has a central role in the neuropathology of Alzheimers disease. Region specific, extracellular accumulation of Aβ peptide is accompanied by microgliosis, cytoskeletal changes, dystrophic neuritis and synaptic loss. These pathological alterations are thought to be linked to the cognitive decline that defines the disease.
Administration of amyloid beta protein or, in particular, Aβ1-28 in amounts of up to 10−2 mg/dose in the absence of any adjuvants and without any linkage of the amyloid beta protein or Aβ1-28 to a carrier, for the treatment of Alzheimer's disease, is described in EP 526'511.
Others have used administration of Aβ peptides in combination with adjuvants, to induce an immune response, cellular or humoral, against Aβ1-42. In a transgenic mouse model of Alzheimer disease, animals overexpress human amyloid precursor protein containing the mutation APP(717)V-F (PDAPP-mice; Johnson-Wood, K. et al., Proc. Natl. Acad. USA 94: 1550-1555, Games, D. et al., Nature 373: 523-527 (1995a)), leading to overproduction of Aβ1-42, develop plaques, dystrophic neuritis, loss of presynaptic terminals, astrocytosis and microgliosis. In a recent study, Schenk, D. et al., (Nature 400:173-77 (1999) and WO 99/27944) report that administration of aggregated Aβ1-42 mixed with a strong adjuvant (CFA/IFA), which cannot be used in humans, in the first 4 immunizations, followed by administration of aggregated Aβ1-42 in PBS in the subsequent immunizations, to PDAPP-mice at 6 weeks of age, essentially prevented plaque formation and associated dystrophic neuritis. The same authors reported that immunization of older mice (11 months of age) using the same strategy markedly reduced the extent and progression of Alzheimer's disease (AD)-like neuropathologies. Proliferation of splenocytes from mice immunized using the aforementioned strategy was reported in Example III (Screen for therapeutic Efficacy against established AD) of WO 99/27944, showing that Aβ1-42 specific T-cells were induced by the vaccination procedure. Coupling of Aβ fragments to sheep anti-mouse IgG, and immunization of said coupled fragment in the presence of the adjuvant CFA/IFA is reported in WO 9927944. The use of compositions comprising Aβ fragments linked to polypeptides such as diphtheria toxin for promoting an immune response against Aβ is also disclosed in WO 99/27944. However, no data of immunization are provided.
A monoclonal antibody recognizing an epitope within the N-terminus (1-16) of Aβ (antibody 6C6) has been shown to protect PC12 cells from neurotoxicity of fibrillar β-amyloid, and to disaggregate β-amyloid in vitro (Solomon B. et al., Proc. Natl. Acad. Sci. USA (1997)). A monoclonal antibody raised against Aβ1-28, was also shown to suppress β-amyloid aggregation in vitro (Solomon B. et al., Proc. Natl. Acad. Sci. USA (1996)). Frenkel et al., (J. Neuroimmunol. 88: 85-90 (1998)) have later identified the epitope of two anti-aggregating antibodies, 10D5 and 6C6, as being the epitope “EFRH”, i.e. Aβ3-6. In contrast, an antibody specific for Aβ1-7 was unable to prevent β-amyloid aggregation (Frenkel D. et al., J. Neuroimmunol. 95: 136-142 (1999)).
Aβ1-42 is fibrillogenic, and indeed, the vaccine composition described in WO 99/27944 used Aβ1-42 treated in such a way that it can form aggregates. It has been shown that those fibrils are toxic for neuronal cell cultures (Yankner et al., Science 245: 417-420 (1989)), and that a toxic effect is also observed when injected into animal brains (Sigurdson et al., Neurobiol. Aging 17: 893-901 (1996); Sigurdson et al., Neurobiol. Aging 18: 591-608 (1997)). Walsh et al., (Nature 416:535-539 (2002)) report that natural oligomers of Aβ are formed within intracellular vesicles. Those oligomers inhibited long term potentiation in rats in vivo and disrupted synaptic plasticity at concentrations found in human brain and cerebrospinal fluid.
In another study, Bard, F. et al. (Nature Medicine 6:916-19 (2000)) reported that peripheral administration of antibodies raised against Aβ1-42, was able to reduce amyloid burden, despite relatively modest serum levels. This study utilized either polyclonal antibodies raised against Aβ1-42, or monoclonal antibodies raised against synthetic fragments derived from different regions of Aβ. Thus induction of antibodies against Aβ peptides bears promises as a potential therapeutic treatment for Alzheimer disease.
Mucosal administration of an antigen associated with β-amyloid plaques, such as β-amyloid peptide and Aβ1-40, has been described in WO99/27949. Mucosal administration is said to suppress certain cytokine responses associated with Alzheimer's disease, and to enhance certain other cytokine responses associated with the suppression of inflammatory responses linked to the disease. It is thought that suppression of the inflammatory responses is effected by the “elicitation of T-cells characterized by an anti-inflammatory cytokine profile”. Suitable antigens, as described in WO9927949, include antigens specific for AD, and which are recognized by immune T-cells of a human or animal host.
Fusion of epitopes of a monoclonal antibody recognizing Aβ to coat proteins of filamentous phages is described in WO 01/18169. Immunization of mice with the filamentous phages displaying the 15-mer epitope VHEPHEFRHVALNPV (SEQ ID NO: 89) on the coat protein VIII resulted in antibodies recognizing Aβ1-16, and Aβ1-40. This was demonstrated in an inhibition ELISA using Aβ peptides, and an IC50 of 1 μM was found for inhibition of the binding of the sera to Aβ1-16 with Aβ1-40. Solomon (WO 01/18169), however, provides no indication that the sera elicited against the filamentous phages carrying the VHEPHEFRHVALNPV epitope (SEQ ID NO: 89), bind to amyloid plaques or neuritic plaques of AD.
There are a number of drawbacks in using sequences differing from the antigen against which an immune response is to be elicited for immunization. First, antibodies against part of the sequence foreign to the antigen or antigenic determinant may be induced. Second, the conformation of the antigen in the context of the foreign flanking sequence element may be different than in the context of the full-length antigen. Thus, although antibodies cross-reacting to the antigen may be elicited, their binding to the antigen may be suboptimal. The fine specificity of those elicited antibody may also not correspond to the specificity of antibodies elicited against the antigen itself, as additional sid-chains different from the residues present on the full-length Aβ are present in the epitope. Finally, a 15-mer amino-acid sequence may contain T-cell epitopes. Display of the epitope YYEFRH (SEQ ID NO: 90) on the protein III of filamentous phage coat, of which 3-5 copies only are usually present on each phage, is also disclosed in WO 01/18169. Several problems arise when using infectious phages as carrier for immunization. First, production of infectious agents in large scale and in sufficient quantity for large immunization campaigns is problematic. Second, the presence of the DNA of the phage containing antibiotic resistance genes in the vaccine is not unproblematic and is a safety issue. Finally, the feasibility and efficacy of irradiation of large quantities of phages, in the case where non-infectious phages are used as vaccine, is unresolved.
Aβ analogues, wherein Aβ is modified to include T helper epitopes have been described (WO 01/62284). Immunization of TgRND8+ mice, transgenic for human APP, with the Aβ analogue resulted in a 4- to 7.5-fold higher antibody titer over immunization with Aβ1-42 in the absence of adjuvant.
Recent studies demonstrated that a vaccination-induced reduction in brain amyloid deposits has the potential to improve cognitive functions (Schenk, D., et al. Nature 400: 173-177 (1999); Janus, C. et al., Nature 408: 979-982 (2000); Morgan, D. et al., Nature 408: 982-985 (2000)).
The autopsy of a patient immunised with aggregated Aβ1-42 in the Adjuvant QS21 has revealed the presence of a T-lymphocyte meningoencephalitis and infiltration of cerebral white matter by macrophages (Nicoll, J. A. et al., Nature Med. 9: 448-452 (2003)).
Recently, a publication has reported 18 cases of meningoencephalitis in patients immunized by the AN1792, a vaccine composed of aggregated Aβ1-42 and QS-21 as adjuvant (Orgogozo J.-M. et al., Neurology 61: 46-54 (2003)). T-cell activation is reported as a potential mechanism responsible for the disease, while there was no clear relation between disease and anti-Aβ1-42 titers in the serum.
It is well established that the administration of purified proteins alone is usually not sufficient to elicit a strong immune response; isolated antigen generally must be given together with helper substances called adjuvants. Within these adjuvants, the administered antigen is protected against rapid degradation, and the adjuvant provides an extended release of a low level of antigen. In the present invention, Aβ peptides are made immunogenic through binding to a VLP and do not require an adjuvant.
One way to improve the efficiency of vaccination is thus to increase the degree of repetitiveness of the antigen applied. Unlike isolated proteins, viruses induce prompt and efficient immune responses in the absence of any adjuvants both with and without T-cell help (Bachmann and Zinkernagel, Ann. Rev. Immunol: 15:235-270 (1991)). Although viruses often consist of few proteins, they are able to trigger much stronger immune responses than their isolated components. For B-cell responses, it is known that one crucial factor for the immunogenicity of viruses is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasi-crystalline surface that displays a regular array of epitopes which efficiently crosslinks epitope-specific immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol. Today 17:553-558 (1996)). This crosslinking of surface immunoglobulins on B cells is a strong activation signal that directly induces cell-cycle progression and the production of 1 gM antibodies. Further, such triggered B cells are able to activate T helper cells, which in turn induce a switch from IgM to IgG antibody production in B cells and the generation of long-lived B cell memory—the goal of any vaccination (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)). Viral structure is even linked to the generation of anti-antibodies in autoimmune disease and as a part of the natural response to pathogens (see Fehr, T., et al., J Exp. Med. 185:1785-1792 (1997)). Thus, antibodies presented by a highly organized viral surface are able to induce strong anti-antibody responses.
As indicated, however, the immune system usually fails to produce antibodies against self-derived structures. For soluble antigens present at low concentrations, this is due to tolerance at the Th cell level. Under these conditions, coupling the self-antigen to a carrier that can deliver T help may break tolerance. For soluble proteins present at high concentrations or membrane proteins at low concentration, B and Th cells may be tolerant. However, B cell tolerance may be reversible (anergy) and can be broken by administration of the antigen in a highly organized fashion coupled to a foreign carrier (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)). As shown in pending U.S. application Ser. No. 10/050,902 filed on Jan. 18, 2002, strong immune responses could be induced with compositions comprising Aβ peptides (Aβ1-15, Aβ1-27 and Aβ33-42, which is a self-antigen in mice) bound to a VLP. In particular, the aforementioned human Aβ peptides bound to the VLP of RNA phage Qβ induced high Aβ specific titers in human APP transgenic mice (described in Example) demonstrating that tolerance to the self-antigen Aβ could be overcome by immunizing with Aβ peptides bound to a VLP.
There is thus a need for highly immunogenic safe compositions and vaccines, respectively, to treat Alzheimer diseases, in particular, using immunogens devoid of T-cell epitopes and adjuvants, respectively, which might elicit side-effects, and still being capable of inducing high antibody titers, which antibodies, furthermore, being capable of binding to amyloid plaques.